Vertebrate cardiac and striated muscle contraction has been hypothesized to be regulated by altering either the number of myosin-binding sites on actin or the kinetics of the force generating reaction. In addition, this regulation has also been hypothesized to be modulated not just by the primary effector, Ca2+ binding to troponin C (TnC), but also in turn by crossbridge binding to the thin filament. Our recent experimental observations have led to a new and simpler model to explain the Ca2+ regulation of actomyosin binding and kinetics (outlined in the Introduction): that the molecular dynamics of thin filament regulatory units directly influence the apparent kinetics of actomyosin function at submaximal activation. In Project II, we will use permeabilized preparations to examine the molecular regulatory mechanism of dynamic processes during muscle contraction as indicated by three mechanical kinetic parameters: the rate of isometric tension redevelopment (k/TR); unloaded shortening velocity (V/US); and the myosin 'power stroke,' assessed by phase 2 of tension transients in response to small amplitude length steps. Three chemomechanical parameters have been chosen to test the regulatory mechanism in addition to steady state isometric force to examine on multiple facets of the crossbridge cycle and because each of these parameters exhibits an apparent variation with thin filament activation level, insofar as they have been examined. The first specific hypothesis to be tested is that k/TR at submaximal Ca2+-activation reflects dynamic properties of individual regulatory units. Directly related to this hypothesis, as supported by our Preliminary data, is that the type of TnC in a regulatory unit is a major determinant of the dynamics of that unit during submaximal activations. The second hypothesis is that the kinetics of the myosin unitary 'power stroke,' as reflected by millisecond time scale tension transients following a step change in length, are regulated by Ca2+. At the very least, these experiments will allow us to distinguish between hypotheses that phase 2 kinetics are modulated by either (a) a transient population of an initial attached, low force producing actomyosin intermediate, vs. (b) cooperative interactions between crossbridges such that the back rate constant of the force-producing transition decreases as the fraction of attached crossbridges increases. The third hypothesis is that unloaded shortening velocity is primarily regulated by different mechanisms in cardiac vs. skeletal muscle. These experiments using skinned fiber preparations will be complemented with measurements of filament sliding velocity in Project IV using in vitro motility assays on purified proteins. In total, we will be testing the validity and universality of a new hypothesis--that the dynamic properties of individual regulatory units play a major and heretofore unrecognized role in determining the macroscopic kinetics of muscle contraction.